Functional & Integrative Genomics

https://doi.org/10.1007/s10142-020-00736-x

ORIGINAL ARTICLE

Bacillus subtilis EA-CB0575 genome reveals clues for plant growth

promotion and potential for sustainable agriculture
Nicolás D. Franco-Sierra 1 & Luisa F. Posada 1,2 & Germán Santa-María 1 & Magally Romero-Tabarez 2 &
Valeska Villegas-Escobar 3 & Javier C. Álvarez 3

Received: 11 November 2019 / Revised: 17 January 2020 / Accepted: 21 February 2020

# Springer-Verlag GmbH Germany, part of Springer Nature 2020

Abstract
Bacillus subtilis is a remarkably diverse bacterial species that displays many ecological functions. Given its genomic diversity,
the strain Bacillus subtilis EA-CB0575, isolated from the rhizosphere of a banana plant, was sequenced and assembled to
determine the genomic potential associated with its plant growth promotion potential. The genome was sequenced by Illumina
technology and assembled using Velvet 1.2.10, resulting in a whole genome of 4.09 Mb with 4332 genes. Genes involved in the
production of indoles, siderophores, lipopeptides, volatile compounds, phytase, bacilibactin, and nitrogenase were predicted by
gene annotation or by metabolic pathway prediction by RAST. These potential traits were determined using in vitro biochemical
tests, finding that B. subtilis EA-CB0575 produces two families of lipopeptides (surfactin and fengycin), solubilizes phosphate,
fixes nitrogen, and produces indole and siderophores compounds. Finally, strain EA-CB0575 increased 34.60% the total dry
weight (TDW) of tomato plants with respect to non-inoculated plants at greenhouse level. These results suggest that the
identification of strain-specific genes and predicted metabolic pathways might explain the strain potential to promote plant
growth by several mechanisms of action, accelerating the development of plant biostimulants for sustainable agricultural.

Keywords Bacillus . Plant growth-promoting rhizobacteria (PGPR) . Biostimulants . Genomic and biochemical potential

Introduction mechanisms like phytohormone production, nitrogen fixation,

siderophore, antibiotic production, induced systemic resis-
Plant growth-promoting rhizobacteria (PGPR) have been used tance (ISR), and competition between others (Backer et al.
as biostimulants and biopesticides in different crops of eco- 2018). From the commercially available PGPRs, Bacillus
nomic relevance. They promote plant growth through diverse subtilis outstand for its resistance to stress conditions due to

Nicolás D. Franco-Sierra and Luisa F. Posada contributed equally to this

work.
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s10142-020-00736-x) contains supplementary
material, which is available to authorized users.

* Javier C. Álvarez Valeska Villegas-Escobar

jcorre38@eafit.edu.co vvilleg2@eafit.edu.co
Nicolás D. Franco-Sierra
1
nfranco@eafit.edu.co Department of Process Engineering, Research group CIBIOP,
Universidad EAFIT, Cra. 49 # 7 Sur-50, Medellín, Antioquia,
Luisa F. Posada Colombia
lposadau@eafit.edu.co 2
Universidad Nacional de Colombia, Carrera 65 # 59A - 110,
Germán Santa-María Medellín, Antioquia, Colombia
gsantam@eafit.edu.co 3
Department of Biological Sciences, Research group CIBIOP,
Magally Romero-Tabarez Universidad EAFIT, Cra. 49 # 7 Sur-50, Medellín, Antioquia,
mromerota@unal.edu.co Colombia

spore formation, broad metabolite spectrum, fast growth in
liquid media, and the ability to colonize plant surfaces
(Kumar et al. 2011). Therefore, different efforts have been
made to elucidate its potential and mechanisms of action from
different omics perspectives.
From the genome perspective, the first sequenced strain
was B. subtilis Marbug 168, which has also become the most
studied Gram-positive bacterium, revealing important infor-
mation about sporulation, germination, nutrition, and devel-
opment for B. subtilis group. Its genome is 4.2 Mb composed
of 4100 genes that encode proteins from which 4% is devoted
to antibiotic production and another 4% for sporulation and
germination processes (Kunst et al. 1997; Moszer 1998).
Since then, the whole-genome sequence of around 200 strains
of B. subtilis group have been sequenced (https://www.ncbi.
nlm.nih.gov/assembly/?term=Bacillus%20subtilis),
generating information on the genomic diversity but also on
the systematics of Bacillus genus and the adaptations of this
taxon in their natural habitat (Li et al. 2016; Smith et al. 2014;
Zeigler 2011). Access to whole-genome sequences and their
comparative analyses with different Bacillus subtilis strains
and close relatives is important to understand gene flow, spe-
ciation, diversity, and genomic dynamics in bacilli for physi-
ological, ecological, and evolutive studies (Earl et al. 2012;
Shaligram et al. 2016). Currently, there are studies where sev-
eral genomes of Bacillus strains are elucidated in order to
establish the safety and the efficiency for industrial microbi-
ology (Sulthana et al. 2019), to investigate the genetics of
fermenting strains and its relationship with the productivity
of metabolites (Deng et al. 2019; Kamada et al. 2015), to
clarify the phylogeny of Bacillus groups (Stevens et al.
2019), or to determine plasmid-independent species-species
markers (Ivanova et al. 2003). The study of the genome of
microorganisms used for biofertilizers and biostimulants pro-
duction is important to bioinoculants technology because it
helps identify genes that contribute to the beneficial activity
and increasing knowledge of the molecular mechanisms relat-
ed to plant growth potential. Also, this provides information
for inoculants biosafety and the compliance with existing and
developing regulations for this kind of products whose supply
and consumption is growing (EPA 2019). The use of next
generation sequencing (NGS) has allowed whole-genome se-
quencing of PGPRs (plant growth-promoting rhizobacteria)
isolated from different crops such as wheat, pepper, cotton,
and coconut (Gupta et al. 2014; Kai-Jium et al. 2018); and it is
an opportunity to obtain information about strains used and to
design strategies for development and use of such PGPRs to
support sustainable agriculture.
In this research, we present the sequencing, assembling,
and characterization of B. subtilis EA-CB0575 whole ge-
nome, isolated from a commercial crop of Musa AAA in
Colombia; this microorganism has shown successful results
for plant growth promotion of banana crops, maize, and
Funct Integr Genomics
tomato (Posada et al. 2018). We hypothesize that the genome
sequence of B. subtilis EA-CB0575 will reveal genes involved
in plant growth promotion mechanisms, which will be
expressed in vitro and potentially promote growth of tomato
plants. Therefore, the presence of homologous genes associ-
ated with mechanisms of plant growth promotion such as
indole and siderophores production, phosphate solubilization,
nitrogen fixation, LPs production, and production of volatile
compounds was determined for the assembled genome.
Biochemical traits, related to these characteristics, were eval-
uated at in vitro level, and the strain’s potential for growth
promotion of tomato crop was determined at greenhouse level
as evidence of the relationship between the genomic potential
and the capacity of growth promotion in a plant.
Methodology
Microorganisms and culture conditions
B. subtilis EA-CB0575 was isolated from roots of a banana
plant cv. Valery from Urabá – Colombia (Northeastern 07° 51′
58.6″ N, − 76° 37′ 39″ W) in 2009 (Posada et al. 2016). The
microorganism was identified by analysis of 16s rRNA gene
sequencing (Accession number KC170988) and characterized
as a Gram-positive rod (1.85 ± 0.31 μm), forming central
spores (0.72 ± 0.15 μm), and producing irregular and matt
white colonies with crateriform elevation in TSA X 0.5
(105458, Merck). The strain was stored in TSB medium
(TSB, 105459, Merck) with glycerol (20% v/v) at − 80 °C
and registered on the bacterial collection RNC 191 (Instituto
Alexander Von Humboldt). B. subtilis 168 (Zeigler et al.
2008) and B. velezensis FZB42 (Fan et al. 2018), previously
B. amyloliquefaciens FZB42 (Fan et al. 2012), were donated
by Dr. Camilo Ramírez (Universidad de Antioquia,
Colombia) and Dr. Rainer Borriss (ABiTEP GmbH ABI,
Germany) respectively.
Genomic DNA preparation
B. subtilis EA-CB0575 genomic DNA was obtained from
cells harvested in the exponential phase of a submerged cul-
ture in TSB. Briefly, the strain was cultured 14 h at 150 rpm
and 30 °C, centrifugated, and obtained pellet used for DNA
extraction using Ultraclean Microbial DNA isolation (Mobio).
DNA quality was determined using NanoDrop and agarose
1% gel electrophoresis.
Sequencing and annotation of B. subtilis EA-CB0575
genome
B. subtilis EA-CB0575 genome was sequenced using Illumina
HiSeq 2000 paired-end method and 100 bp reads with 265 bp
Funct Integr Genomics
average insert size were obtained. The quality of the FASTQ
files was verified with FastQC (Andrews 2010) and reads
were trimmed to ensure high quality (Phred score > 30) using
Trimmomatic version 0.35 (Bolger et al. 2014). The genome
was assembled with Velvet 1.2.10 (Zerbino and Birney 2008)
using k-mer = 95 and derived statistics of the assembly pro-
cess were determined (genome total length, N50, N90, time for
running (min), cluster numbers, cluster average size (pb)).
Subsequently, OriFind and Rast software (Aziz et al. 2008)
were used for ORI and ORF prediction, respectively, and the
order of alignments with ProgressiveMauve aligner and
Murasaki software (Popendorf et al. 2010) with B. subtilis
168 as the reference strain. Once the genome was assembled,
gene prediction and annotation were performed using RAST
(http://rast.nmpdr.org/), and circular genome representation
was done with CGView webserver (http://stothard.afns.
ualberta.ca/cgview_server/). RAST metabolic predictions
were used to determine indole, siderophores, and volatile
compounds production. Phosphate solubilization and
nitrogen fixation capacity were determined to evaluate the
presence of phy and nif genes inside the genome. This
search was done using the reference strains B. velezensis
FZB42 (GenBank Accession number: CP000560.1) for
indole, specifically IAA (indole acetic acid), volatiles, and
siderophores production; B. subtilis 168 for phytase
production; and Klebsiella pneumoniae (Genbank accession
number X13303.1) for nitrogen fixation. Local BLAST
between references and B. subtilis EA-CB0575 was per-
formed and subsequently, a combined procedure between
the RAST metabolic annotation (KEGG tool) and the search
for genes of interest was used to find related genes to the
PGPR traits in the assembled genome of the B. subtilis strain
EA-CB0575. The nif and phy cluster genes search was per-
formed comparing the homology of these genes with sections
of the evaluated strains.
Identification and characterization of NRPS genomic
clusters
The presence of non-ribosomal peptide synthetases (NRPS)
coding regions in B. subtilis EA-CB0575 genome was evalu-
ated using homologous sequences to coding regions of
B. velezensis FZB42 strain, which produce three families of
LPs (Koumoutsi et al. 2004). BLASTn algorithm was used to
determine differences in operon sequences throughout the ge-
nome, and Murasaki software 1.68.6 (Popendorf et al. 2010)
was used to determine ORFs. RAST and FgenesB operon
predictor (Softberry, Inc., NY, USA) was used to evaluate
coding sequences. NCBI Conserved Domain Database
(CDD) (Marchler-Bauer et al. 2011) was used to contrast re-
ported information with B. subtilis EA-CB0575 genome in-
formation and to predict structural domains related to each
ORF previously found.
Comparative genomics of B. subtilis EA-CB0575
and phylogenetic study
B. velezensis FZB42, B. subtilis 168, and B. subtilis EA-
CB0575 genomes were compared to locate gene clusters in-
volved in the secondary metabolites production using
Murasaki 1.68.6 software (Popendorf et al. 2010) and GMV
tool. A phylogenetic reconstruction analysis was performed to
infer the evolutionary relationships of B. subtilis EA-CB0575
and others Bacillus spp. with whole genomes reported in
GenBank NCBI. This analysis was done using five house-
keeping genes (rpoB, gyrA, purH, polC, and groEL) for
thirty-one Bacillus spp. strains and using Peptoclostridium
difficile as the outgroup (Table 1). Bayesian inference (BI)
and maximum likelihood (ML) were used for the phylogenetic
reconstruction. Sampled genes were aligned independently in
an amino acid–based fashion using RevTrans. First, nucleo-
tide coding sequences were translated to amino acids using
VirtualRibosome from RevTrans 1.4 package and using ge-
netic bacterial code table # 11 (https://www.ncbi.nlm.nih.gov/
Taxonomy/Utils/wprintgc.cgi). Amino acid sequences of each
gene were aligned independently using MAFFT 7.213 in
automatic mode. Then, alignments were reverse-translated
from amino acids to nucleotides with RevTrans 1.4 software
(Wernersson and Pedersen 2003). Aligned nucleotide se-
quences for each gene were concatenated into a single
FASTA file and converted to NEXUS format by ReadSeq 2.
1.19 (Gilbert 2003), and to Phylip using Biopython utilities.
Partitionfinder 1.1 software (Lanfear et al. 2014) was used to
find the best partitioning scheme for the data and the appro-
priate nucleotide substitution models for each partition. The
BI analysis was done using the software MrBayes 3.2.2
(Ronquist et al. 2012). Four independent MCMC (Markov
Chain Monte Carlo) analyses were carried out with 10 million
generations and eight chains (seven hot and one cold) for each
one and a relative burn-in fraction of 0.35. Consensus tree for
BI was obtained using the remaining trees. ML analysis was
performed with Garli HPC-MPI 2.07 (Bazinet et al. 2014). A
search was carried out with eight replicates and the one with
the highest probability was selected (likelihood), followed by
an analysis with 1000 bootstrap replicates. SumTrees 3.3.1
(Sukumaran and Holder 2010) was used to map bootstrap
replicates on the best tree from the first analysis. The visual-
ization of the obtained trees was done with FigTree 1.4.1
(Rambaut 2014). The roots of both trees were fixed based
on the external group used (Peptoclostridium difficile).
Phenotypic characterization of B. subtilis EA-CB0575
and reference strains
Production of total indoles was determined by the colorimetric
technique using Salkowski’s reagent as described by Patten
and Glick (2002). Siderophore production was determined by
 Funct Integr Genomics

Table 1 Microorganisms used for

the phylogenetic analysis Accession number Strain Reference

NC_006270.3 Bacillus licheniformis DSM 13 ATCC 14580 Veith et al. 2004

NZ_CP010524.1 Bacillus licheniformis BL-09 Pengfei et al. 2015
NC_009848.1 Bacillus pumilus SAFR-032 Tirumalai et al. 2013
CP011150.1 Bacillus pumilus W3 Zheng-Bing et al. 2015
NC_012659.1 Bacillus anthracis str. A0248 Md. Anisur et al. 2014
NC_005957.1 Bacillus thuringiensis serovar konkukian str. 97-26 Li et al. 2015
NZ_CP009335.1 Bacillus thuringiensis HD1011 Li et al. 2015
NC_004722.1 Bacillus cereus ATCC 14579 Ivanova et al. 2003
NC_011969.1 Bacillus cereus Q1 Zhaohui et al. 2009
NC_012472.1 Bacillus cereus 03BB102 Costa et al. 2018
NZ_CP009692.1 Bacillus mycoides ATCC 6462 Li et al. 2018
NZ_CP007626.1 Bacillus mycoides 219,298 Liu et al. 2017
CP009746,1 Bacillus weihenstephanensis WSBC 10204 Liu et al. 2018
CP006952.1 Bacillus amyloliquefaciens LFB112 Cai et al. 2014
NC_022653.1 Bacillus amyloliquefaciens CC178 Kim et al. 2015
NC_014551.1 Bacillus amyloliquefaciens DSM 7 Rückert et al. 2011
CP000560.1 Bacillus velezensis str. FZB42 Chen et al. 2007
NC_016047.1 Bacillus subtilis subsp. spizizenii TU-B-10 Zeigler 2011
CP002183.1 Bacillus subtilis subsp. spizizenii str. W23 Zeigler 2011
NZ_CM000487.1 Bacillus subtilis subsp. subtilis str. 168 Kunst et al. 1997
NZ_CM000489.1 Bacillus subtilis subsp. subtilis str. JH642 Smith et al. 2014
NZ_CM000488.1 Bacillus subtilis subsp. subtilis str. NCIB 3610 Nye et al. 2017
NZ_CM000490.1 Bacillus subtilis subsp. subtilis str. SMY Zeigler et al. 2008
NC_020507.1 Bacillus subtilis subsp. subtilis 6051-HGW Khatri et al. 2016
NC_020832.1 Bacillus subtilis subsp. subtilis str. BAB-1 Guo et al. 2015
NC_017195.1 Bacillus subtilis subsp. subtilis str. RO-NN-1 Zeigler 2011
NC_020244.1 Bacillus subtilis XF-1 Guo et al. 2015
CP002468.1 Bacillus subtilis BSn5 Deng et al. 2011
CP006881.1 Bacillus subtilis PY79 Schroeder and Simmons 2013
KC170988 Bacillus subtilis EA-CB0575 This study
GQ375229 Bacillus subtilis EA-CB0015 Unpublished
NZ_LN614756.1 Peptoclostridium difficile CD630DERM Pereira et al. 2016

the colorimetric CAS method (Schwyn and Neilands 1987);
phosphate solubilization by growing on NBRIP medium
(NaCl 1 g; CaCl 2 *2H 2 O 0.2 g; MgSO 4 *7H 2 O 0.4 g;
NH4NO3 1 g; glucose 10 g, benomyl 300 ppm, bactoagar
7 g, and phosphoric rock 3.5 g, using phosphoric rock instead
tricalcium phosphate) (Bashan et al. 2013; Kim et al. 1997;
Murphy and Riley 1962) and nitrogen fixation were deter-
mined by growth on NFb medium (Eckert et al. 2001).
Antagonistic potential against banana pathogens as
Fusarium oxysporum (strain EAHP-0015, donated by
Banana Growers Association from Colombia, AUGURA)
and Ralstonia solanacearum (EA-EP009, isolated of banana
plant at Universidad EAFIT, Medellin) was evaluated by
using the dual-culture plate method previously described by
Lemessa and Zeller (2007). Antagonistic potential against
Pseudocercospora fijiensis (isolated of banana leaves plant
using necrotic tissue by ascospore discharge method) was
evaluated using the varnish technique modified by Talavera
et al. (1998) and percent inhibition of germ tube was deter-
mined by Gutierrez-Monsalve et al. (2015). Finally, produc-
tion and purification of lipopeptides was carried out by a
methodology previously described (Villegas-Escobar et al.
2013). Briefly, 20 mL of an overnight culture of B. subtilis
EA-CB0575 (12 h, 30 °C) was transferred to 500-mL
Erlenmeyer flask containing 180 mL of MOLP medium
(Jacques et al. 1999) and incubated for 3 days at 30 °C and
150 rpm. After 12 h of incubation, 4% (w/v) of amberlite resin
XAD-16 (Alfa Aesar®) was added to the culture. After
Funct Integr Genomics
incubation, the resin was recovered, washed with 400 mL of
sterile distilled water, and adsorbed metabolites were eluted
with 200 mL of methanol. The methanolic extract was evap-
orated under reduced pressure in a rotary evaporator (50 °C, −
50 psig) obtaining 99 mg of solid residue from 200 mL total
culture. The solid residue was suspended in distilled water
(37 mg/mL) and applied to a solid-phase extraction (SPE
C18) cartridge (Baker®, 10 g). The cartridge was then rinsed
successively with 80 mL of distilled water, 80 mL of 50%
methanol, and 160 mL of 100% methanol. The methanolic
fraction was evaporated and the solid residue dissolved in
methanol (50 mg/mL) for RP-HPLC analysis. The methanolic
fraction was purified by RP-HPLC using an Eclipse XDB C18
column (250 × 4.6 mm, 5 μm) connected to an Agilent
G1311A quaternary pump and with a solvent system
consisting of 0.1% in water (solvent A) and 0.1% TFA in
acetonitrile (solvent B). Forty microliters of the sample was
injected into the column and the compounds were eluted by a
gradient program (30/100/100% B in 0/25/35 min) at a flow
rate of 1 mL/min and UV detection at 214 nm. Peaks with
different retention times were collected, vacuum evaporated
(Eppendorf Concentrator Plus™, Harburg, Germany), and
stored at 4 °C. Purified HPLC fractions and surfactin standard
(Sigma Aldrich, S3523) were analyzed by high-resolution
mass spectrometry (Iowa State University’s Protein Facility,
Ames, IO, USA) using an HPLC (Agilent 1260) coupled to
Thermo Scientific Hybrid Q-Exactive ™ quadrupole-orbitrap
tandem mass spectrometer equipped with a NanoSpray Flex
ionization source and cyano cinnamic acid as matrix. Solvent
A (water + 0.1% formic acid) and B (acetonitrile with 0.1%
formic acid) with a gradient of 20–100% were used for
13 min. Data were acquired in a range between 200 and
2000 m/z in positive ionization mode. MS/MS analysis was
performed using an HCD ion collision cell and the software
Xcalibur (Thermo Scientific, version 2.2). Proteowizard
3.0.10.200 (Chambers et al. 2012) was used for the analysis
of the results.
Plant growth promotion of B. subtilis EA-CB0575
in tomato
Plant growth promotion effect of B. subtilis EA-CB0575 and
two reference strains (B. velezensis FZB42 and B. subtilis 168)
were evaluated in tomato variety Chonto-Santa Cruz at green-
house level using a completely randomized design (CRD).
Tomato seeds were disinfected successively with 70% ethanol
for 15 min, 5% sodium hypochlorite for 5 min, and three
washes with sterile distilled water. After scarification at 4 °C
for 24 h, seeds were inoculated by immersing the roots in a
bacterial suspension (1 × 108 CFU/mL) or in sterile distilled
water (control) for 1 h. The seeds were sowed in pots with
500 g of commercial soil (1 sand: 0.5 black soil: 0.5 rice husk)
and kept at greenhouse conditions (22–30 °C, 12 h light/12 h
dark, 60% of maximum water-holding capacity) for 1 month.
Plants were fertilized after 15 days of inoculation with 15-15-
15 NPK. The growth-promoting effect of the different strains
was performed by measuring shoot length (SL) and dry
weights (shoot dry weight (SDW), root dry weight (RDW),
and total dry weight (TDW)). This experiment was conducted
twice with 8 replicates per treatment and two plants per pot.
Statistical analysis
Analysis of variance (ANOVA) was used to analyze each
experiment in RStudio. The assumptions of normality
(Shapiro-Wilks test), homoscedasticity (Levenne’s test and
Barlett’s test), and independence (graphic residues vs. run or-
der and Durbin-Watson statistics) were determined. In the case
of significant P value (P < 0.05), means were compared by
using LSD or Dunnett multiple comparison test.
Results
Genome analysis of B. subtilis subsp. subtilis
EA-CB0575 reveals the potential for plant growth
promotion
16S rRNA gene sequencing was performed to define the strain
taxonomy, finding that it belongs to Bacillus subtilis species
with a query cover of 100%, an identity of 99%, and an E-
value of 0.0. The complete genome of B. subtilis EA-CB0575
was assembled in 16 contigs, reaching an average size of
255,665 bp. For this assembly, an average G-C content of
43.7% and a total genome size of 4.09 Mb was determined
(Fig 1). The average coverage of the cluster was 110× and half
of the genome bases were found in 2 contigs (N50), 90% in 6
(N90), and 95% in 7 contigs (N95). The maximum coverage
of the genome was 2000×. Table 2 shows the number of genes
annotated and involved in B. subtilis EA-CB0575 metabolic
and cellular processes. There is a high similarity among the
number of genes in each category between the strain EA-
CB0575 and B. subtilis 168 type strain; but the former has
more genes in several metabolism-related functions (fatty
acids, lipids and isoprenoids, sulfur, carbohydrates, aromatic
compounds, and phosphorus) and in cellular processes (divi-
sion and cellular cycle, cell wall formation and capsule forma-
tion, motility and chemostasis, regulation and cellular signal-
ing, and genes related to phages, prophages, transposable el-
ements, plasmids). The genome contains 4332 genes, 4237
genes encoding proteins, 95 RNA genes (including 10 copies
of the 16S rRNA), 2917 genes encoding proteins with predict-
ed function, and 1320 genes encoding hypothetical proteins.
The presence of related genes to PGPR mechanisms or the
metabolic pathway prediction of RAST was found from the
gene annotation. The production of enzymes involved in the

Fig. 1 Whole-genome
distribution of B. subtilis EA-
CB0575
metabolism of indole via the tryptophan pathway was predict-
ed, suggesting that B. subtilis EA-CB0575 has the potential to
produce these compounds. Indole, indole acetate, and indole-
acetamide production was predicted (Fig. S1). The potential
for production of siderophores as bacillibactin type,
enterochelins (enterobacins), vibriobacins, and related ones
synthesized by NPRSs was found. Some enzymes related to
the production of compounds such as salicylate, isocorismate,
and dihydroxybenzoate would likely be produced by
B. subtilis EA-CB0575 (Fig. S2). The gene cluster that en-
codes to produce bacilibactin (dhbACEBF) was found in the
strain genome when compared with the reference genomes
(Table 3). Volatile compounds as 2,3-butanediol and acetoin
might be produced by this strain (Fig. S3) given that it has the
potential to produce the enzymes α−acetolactate synthetase,
α−acetolactate decarboxylase, acetoin-reductase,
acetylacetoin-synthase, and others reported (Qi et al. 2014).
The phyC genes, coding for enzymes that sequentially remove
phosphates of phytate, were found in the genome of the strain
B. subtilis EA-CB0575, suggesting the capacity of this strain
for organic phosphate solubilization. Also, some genes related
to enzyme activity to fully perform citric acid pathway (TCA
cycle) were found in the strain 575 genome, indicating a pos-
sible inorganic phosphate solubilization by organic acid pro-
duction (Vyas and Gulati 2009).
Finally, the environmental nitrogen fixation capacity of
strain EA-CB0575 was evaluated. The genes nifU, nifS,
Funct Integr Genomics
nifV, and nifF were present in the strain genome which are
involved in nitrogenase enzymatic activity responsible for
the biological fixation of nitrogen. However, the presence of
all the genes of the nitrogenase cluster which are essential for
the nitrogenase activity was not found. The assembled and
annotated genome was saved on Universidad EAFIT server,
operating under Ubuntu 14.04 and accessed through the
GBrowse2 genome browser (Stein et al. 2002). An analysis
of the cluster ends was performed, finding that 50% of the
genes in this section correspond to rRNA, 26.67% to non-
ribosomal peptide synthases, followed by 6.67% for tRNAs,
and the rest corresponds to phages and repeated GXT proteins.
The genome of B. subtilis EA-CB0575 harbors an array
of gene clusters involved in synthesis of secondary
metabolites
The presence of NRPS coding sequences involved in the
synthesis of surfactins, fengycins, and iturins in B. subtilis
EA-CB0575 genome was evaluated using B. velezensis
FZB42 as the genome reference. Coding regions for
surfactins and fengycins production were found; however,
no coding regions were found for iturins production (Fig. 2).
The coding regions for NRPSs that produce surfactins were
found in clusters 5, 6, and 7 and for fengycins production in
clusters 9, 10, and 11. B. subtilis EA-CB0575 loci for NRPS
was compared respect to the same loci for references
Funct Integr Genomics

Table 2 Annotation of genes

involved in the metabolism and Genes function B. subtilis EA-CB0575 B. subtilis 168
other cellular processes of
B. subtilis EA-CB0575 and Genes related to Fatty acids, lipids, and isoprenoids 119 102
strains reference B. subtilis 168 metabolism Amino acids derivatives 428 432
and B. velezensis FZB42
Sulfur 47 44
Carbohydrates 514 504
Cofactors, vitamins, 218 265
prosthetic groups, pigments
Aromatic compounds 13 10
DNA 112 136
Phosphorus 30 25
Iron 28 32
Secondary metabolism 4 6
Nitrogen- proteins 242 269
Nucleosides and nucleotides 122 139
Potassium 15 26
RNA 163 194
Genes related to Division and cellular cycle 55 48
cellular processes Dormancy and sporulation 122 141
Cellular wall and capsule formation 151 130
Photosynthesis 0 0
Miscellaneous 49 64
Motility and chemostasis 89 66
Regulation and cellular signaling 61 50
Related to phages, prophages, 13 0
transposable elements, plasmids
Respiration 69 82
Response to stress 109 111
Membrane transport 73 75
Virulence, disease, and defense 71 77

B. velezensis FZB42 and B. subtilis 168 and 4 ORFs were few nucleotides between assembly clusters 5 and 6, and 6
reconstructed for the surfactin operon, with two gaps of a and 7 in the srfAA and srfAC genes, respectively. Seven

Table 3 Comparative genomics of B. subtilis EA-CB0575 with the reference strains

Compound Type Number Genes Gen B. subtilis B. subtilis B. velezensis

of (kpb) EA- 168 FZB42
genes CB0575

Bacilibactin Siderophore NRPS 5 dhbACEBF 11.7 + + +

Iturin LP NRPS/PKS 4 bmyDABC 37.2 – – +
Bacillomycin
Fengycin NRPS 5 fenABCDE 37.6 + + +
Surfactin NRPS 4 srfABCD 26.2 + – +
Bacilysin Antibiotic NRPS 5 bacABCDE 4.7 + + +
Macrolactin PKS 9 mlnABCDEFGHI 53.9 – – +
Bacillaene PKS 14 baeBCDEGHIJLMNRS. 72.4 + + +
acpK
Difficidine PKS 15 dfnAYXBCDEFGHIJKLM 70.0 – – +
Subtilosin Sactipeptide 8 n/d 6.9 + + –
Dedicated portion of the genome for secondary metabolite production (%) 3.90 3.79 8.02
Genome length (Mpb) 4.09 4.21 3.91
Funct Integr Genomics
 Funct Integr Genomics
ƒFig. 2 Determination of the loci to produce the NRPSs of surfactins,
fengycins, and iturins in B. velezensis FZB42, B. subtilis EA-CB0575,
and B. subtilis 168 genomes. a Diagram of location for responsible loci to
the synthesis of NRPS related to surfactin in B. subtilis EA-CB0575 strain
in contrast to the reference strains B. subtilis 168 and B. velezensis FZB42
b Diagram of location for responsible loci for genes involved in the
production of the iturin bacillomycin and fengycin B in B. subtilis EA-
CB0575 strain in contrast to the reference strains B. subtilis 168 and
B. velezensis FZB42
domains of adenylation and a thioesterase/acyltransferase
were obtained from the amino acid sequences of the
ORFs. Three of 5 ORFs of the operon for fengycin produc-
tion were obtained, corresponding to fenA, fenC, and fenE
genes. Two domains of adenylation were obtained for the
first, two for the second, and one for the adenylation and one
thioesterase for the third. For the reference strains, the cod-
ing regions for the synthases related to the production of
surfactins and fengycins were found; however, the region
of iturin was determined for FZB42 strain and not for 168
strain. Comparative genomics of B. subtilis EA-CB0575
was performed with the two reference strains B. subtilis
168 and B. velezensis FZB42 to produce some secondary
metabolites (Table 3). Similarities were observed in genome
sizes and genes. There is a greater similarity between the
percentages of the genome dedicated to the production of
secondary metabolites in B. subtilis strains than B. velezensis,
this latter with the highest percentage of the genome dedicated
to the production of these compounds (8.02%). Some impor-
tant clusters of genes, such as those related to the production
of antibiotics, siderophore bacilibactin, and some LPs, are
shared among the evaluated species.
B. subtilis EA-CB0575 belongs to the clade
of B. subtilis subsp. subtilis
The phylogenetic relationship of B. subtilis EA-CB0575 with
other Bacillus sp. strains was performed by comparing a set of
housekeeping genes (Fig. 3). The best partitioning scheme of
the alignment for the concatenated genes was composed of 6
subsets, with the 15 preliminary partitions given, one for each
position of codons (1, 2, 3) in each of the 5 genes analyzed.
Two major clades were observed: the first containing
B. thuringiensis, B. anthracis, B. cereus, B. mycoides, and
B. weihenstephanensis, which are Bacillus spp. pathogens of
various hosts. The second clade was composed of B. pumilus,
B. licheniformis, B. subtilis, B. amyloliquefaciens, and
B. velezensis.
PGPR traits found in B. subtilis EA-CB0575 genome are
expressed in vitro
To determine if genes related to PGPR traits were function-
al, different in vitro evaluations were performed (Table 4).
Among evaluated strains, B. subtilis EA-CB0575 produced
IAA (15.0 ± 2.0 μg/mL) and siderophores (15.4 ± 1.1 μM),
solubilized phosphate, and had the ability to fix environ-
mental nitrogen while B. subtilis 168 produced 2.0-fold
lower concentrations of IAA (6.9 ± 1.4 μg/mL) and
siderophores (7.6 ± 0.9 μM), and no capacity to solubilized
phosphate or fix nitrogen. On the other hand, B. velezensis
FZB42 produced IAA and siderophores, solubilized phos-
phate, but has no potential to fix nitrogen in vitro.
B. subtilis EA-CB0575 inhibited the growth of the phyto-
pathogens R. solanacearum (halo 1.2 ± 0.3 cm on BGA
medium), F. oxysporum (reduction of growth of 21 ±
0.1% on PDA medium), and P. fijiensis (reduction of
33.1 ± 4.3% on germinative tubes of ascospores) when
Bacillus was evaluated in coculture with the fungi tested
in vitro (Table 4). Finally, to determine if gene clusters
related to NRPS were expressed in B. subtilis EA-
CB0575, different purified fractions obtained by HPLC
were analyzed by mass spectrometry analysis. Two fami-
lies of lipopeptides were identified: surfactins and
fengycins (Table 4; Fig. 4 and supplementary material
Tables S1 and S2 and, Fig. S5, S6, S7, S8). In particular,
the precursor ions observed at m/z [M + H]+ 1008.66,
1022.67, 1036.69 and at m/z [M + Na] + 1030.64,
1044.66, 1058.67, and 1072.69 [M + Na]+ (Fig. S5) were
used for further MS/MS analysis, and results showed prod-
uct ions characteristic of surfactins with different lengths
of fatty acid chains (C12, C13, C14, C15, and C16) and
therefore different molecular weights. For the other puri-
fied fraction, the precursor ions at m/z 1408, 1435.77,
1463.81, 1477.82, 1491.83, and 1505.85 corresponding
to protonated molecules [M + H]+, and m/z 1444, 1460
to sodiated [M + Na]+, and potassium [M + K]+ adducts
respectively (Fig. S7), were also used for further MS/MS
analysis. In general, product ions obtained were character-
istic of fengycins A (m/z 1080.50 and 966.45) and B (m/z
1108.58 and 994.49). These results all together suggest
that genes found in the genome of B. subtilis EA-CB0575
are functional in vitro and may confer potential for promot-
ing plant growth.
B. subtilis is a potential PGPR for tomato crops
Tomato seeds variety Chonto-Santa Cruz were inoculated
with suspensions of B. subtilis EA-CB0575 as spores or
vegetative cells and the reference strains B. subtilis 168
and B. velezensis FZB42 as vegetative cells. We determined
an average increase of 29% for the variable TDW of the
plants with application of B. subtilis strains respect to the
control. B. subtilis EA-CB0575 was the strain with a higher
increase of this variable (34.60%). SDW and RDW shown
differences between B. subtilis EA-CB0575 and reference
strains and the control, respectively; with percentage

Fig. 3 Phylogeny of Bacillus spp. from housekeeping genes (rpoB, gyrA,
purH, polC, and groEL) obtained by Bayesian inference and maximum
likelihood methods. The support values are shown for the internal nodes:
increase related to control of 48.4% for SDW, when vege-
tative cells of B. subtilis EA-CB0575 were applied; and
54.2% for RDW when spores were inoculated.
B. velezensis FZB42 did not show increase with respect to
control samples without application of the microorganism
(Table 5). These results are consistent with assays previous-
ly reported, indicating a possible relation between
B. subtilis EA-CB0575 colonization root potential and plant
growth potential for tomato (Posada et al. 2018).
Funct Integr Genomics
posterior probability (PP) for BI and percentage of bootstrap (BP) for ML.
The study strains are highlighted in red
Discussion
The results of the genomic assembly of B. subtilis EA-
CB0575 are similar to those reported for other genomes of
B. subtilis strains (Guo et al. 2015; Liu et al. 2018; Rahimi
et al. 2018). The genome presented some breaks in the corre-
sponding sections to genes encoding rRNA, tRNAs, non-
ribosomal peptide synthases, inserts of phages, and
transposases; it could be due to the difficulty for the assembler
Funct Integr Genomics

Table 4 PGPR biochemical traits at in vitro level for Bacillus EA-CB0575 and reference strains Bacillus sp.

PGPR trait B. subtilis EA-CB0575 B. subtilis 168 B. velezensis FZB42

IAA (μg/mL) 15.0 ± 2.0 a 6.9 ± 1.4 b 17.7 ± 1.7 a

Siderophores (μM) 15.4 ± 1.1 a 7.6 ± 0.9 b 9.8 ± 0.3 b
Phosphate solubilization (mg/L) 0.1 ± 0.0 a 0.0 ± 0.0 b 0.1 ± 0.0 a
Nitrogen fixation + − −
LP’s production Surfactins. fengycins Not reported Surfactins. fengycins (Fan et al. 2012)
In vitro antagonism Ralstonia solanacearum (cm) + (1.2 ± 0.3) ND ND
Fusarium oxysporum + (32.6 ± 5.2%) ND ND
Pseudocercospora fijiensis + (33.1 ± 4.3%) ND ND

Different letters denote significant differences (P < 0.05) according to multiple ranges of LSD. + denotes growth in NFb medium indicative of possible
nitrogen fixation. − denotes absence of growth in NFb medium. + denotes positive antagonism. − denotes negative results for antagonism test. *Halo of
inhibition and percentage of inhibition determined by dual plate assay **Percentage of inhibition determined by ascospores germinative tube inhibition
methodology. ***Indoles by Salkowski’s colorimetric methodology, siderophores production evaluation by CAS methodology, phosphate solubilization
by molybdate blue method using NBRIP medium with phosphoric rock, nitrogen fixation by growth on Nfb medium, and lipopeptides production by
HPLC, and mass spectrometry evaluation. ND, not determined

to work with repeating sequences (Nederbragt et al. 2010),
because the algorithm of graphs does not solve copies sepa-
rately (Compeau et al. 2011). In addition, regions with mod-
ular structure preserved at the sequence level, such as NRPSs,
are another cut-off point of the assembly, because of the am-
biguity at the ends that is not computationally solved with the
available information. Fragmentation of the genome obtained
(16 clusters, with an average length of 255,665 bp) is good for
genomic-derived analyses. When the obtained number of
contigs is compared with the number for other similar pro-
jects, in our case, there is a smaller division than for other
Bacillus (205 partitions for the type strain, and 390 for
B. natto BEST195, among others) (Kunst et al. 1997;
Ulyanova et al. 2016).
When some genes related to PGPR mechanisms were an-
notated and metabolic predictions were performed, we deter-
mined that B. subtilis EA-CB0575 genome has genes or en-
zymes related to the conversion pathway of tryptophan to
indole, which is consistent with the determined indole produc-
tion (15 μg/mL), concentration reported as not deleterious to
the plant (Barazani and Friedman 1999). Metabolic prediction
was evaluated for siderophores production, finding its meta-
bolic machinery inside the B. subtilis EA-CB0575 genome,
which was biochemically corroborated with an in vitro pro-
duction of 15.4 μM. These compounds give to the plant and
microorganisms possibilities to improve iron assimilation and
are elicitors of systemic resistance in the plant (Aznar and
Dellagi 2015). The presence of nifU, nifS, nifV, and nifF, all
essential for encoding the components of the enzymatic mod-
ules of nitrogenase (Li et al. 2016), was determined. It sug-
gests the possibility of the strain to fix environmental nitrogen,
which was determined experimentally by B. subtilis EA-
CB0575 growth on NFb medium. The phyC genes, coding
for enzymes that sequentially remove phosphates of phytate,
were found in the genome of the strain B. subtilis EA-
CB0575, indicating a possibility for organic phosphate solu-
bilization. It has been reported, about their presence in
B. subtilis 168 (Idriss et al. 2002). Several genes with potential
enzyme activity to fully perform citric acid pathway (TCA
cycle) were found in the strain 575 genome, such intermediate
acids (e.g., oxalic acid, malic acid, citric acid) could be poten-
tially involved in inorganic phosphate solubilization exhibited
by B. subtilis EA-CB0575. This was corroborated in biochem-
ical evaluations for strain EA-CB0575, which could grow in
medium NBRIP with phosphoric rock and showed positive re-
sults using molybdate blue test (Murphy and Riley 1962). The

Fengycins Surfactins
2.789
3.096

mAU
1.973

3.270

1500
14.865
15.016
13.845
11.217

30.575
17.631

1000
16.462

28.431
16.714
12.233
12.868

28.873
26.691
7.838
7.028

500

0
0 5 10 15 20 25 30 min

Fig. 4 Lipopeptides analysis production by HPLC chromatogram for 40 μL (concentration 10 mg/mL), flow 1 mL/min, 30 °C, column eclipse
SPE-100% methanol extract from B. subtilis EA-CB0575 culture in XDB C18 250 × 4.6 mm 5 um. A: water HPLC + 0, 1% TFA. B:
MOLP medium. HPLC operation conditions: 214 nm, injection of CH3CN + 0.1% TFA. Gradient 30/100/100% B in 0/25/35 min

Table 5 Effect of B. subtilis EA-CB0575 and reference strains inoculation on the promotion of tomato growth at the greenhouse level in commercial
substrate
Treatment SL (cm) LN
B. subtilis EA-CB0575 (VC) 13.5 ± 0.5 6.5 ± 0.2
B. subtilis EA-CB0575 (S) 14.1 ± 1.1 5.9 ± 0.4
B. subtilis 168 (VC) 14.2 ± 0.6 5 ± 0.8
B. velezensis FZB42 (VC) 12.1 ± 0.6 6.4 ± 0.4
Control 9.4 ± 1.9 4.8 ± 0.2
LSD test (P value)
Data are averages of two independent evaluations in different times each with n = 8 per treatment. Different letters correspond to treatments with
significant differences according to the LSD test. The asterisks correspond to the treatments with significant differences with the water control by the
Dunnett test, both with 95% confidence. VC, vegetative cells; SC, spore cells; SL, shoot length (cm); LN, leaves number; SDW, shoot dry weight (g);
RDW, root dry weight (g); TDW, total dry weight (g)
type strain 168 did not grow in the medium and did not show
positive results for the test, indicating that phosphate solubiliza-
tion for this strain could be related to organic solubilization
processes, using phytates (Ahmad et al. 2017). In addition, it
was found that B. subtilis EA-CB0575 possesses the machinery
to produce 2,3-butanediol and acetoin, but this was not corrob-
orated at in vitro level.
The characterization of regions for coding to NPRSs of the
LPs was carried out. B. velezensis FZB42 and B. subtilis 168
strains were used as the reference. It was determined that
B. subtilis EA-CB0575 genome has homologous regions to
regions that codify for surfactins and fengycins NRPSs. No
coding regions were found for iturin production, consistent
with experimental results (Villegas-Escobar et al. 2013). LPs
production in MOLP medium was evaluated using HPLC and
mass spectrometry. We determined the production of
surfactins C12 to C16, as well as fengycins A (C14, C15,
C16, C17) and B (C16 and C17). These compounds could
be responsible for antagonistic activity against evaluated phy-
topathogens, as reported by several authors (Fan et al. 2018;
Villegas-Escobar et al. 2013). We determined the presence of
loci for surfactins and fengycins codification for the reference
strains, but their in vitro evaluations were not done. Different
reports indicate lack of detection for these that compounds at
in vitro cultures of the B. subtilis 168 as being due to a muta-
tion in the sfp gene, which affects the production of surfactins
(Chen et al. 2007). Loci to encode NRPS production of iturins
was found for FBZ42 strain; and experimental results for re-
ported researches shown in vitro production of the three LP
families announced (Koumoutsi et al. 2004).
B. subtilis EA-CB0575 has more elements inserted in
the genome than the reference strains, possibly because
of their wild character and the domestication of the refer-
ence strain 168 (Veening et al. 2006). In addition, 3.9% of
its genome is associated with secondary metabolism.
However, its potential is related not only to the number
of genes involved but also to the levels being expressed
Funct Integr Genomics
SDW (g) RDW (g) TDW (g)
0.38 ± 0.03 a* 0.13 ± 0.01 ab 0.50 ± 0.03 a*
0.28 ± 0.01 b 0.18 ± 0.01 a* 0.47 ± 0.02 a*
0.33 ± 0.03 ab 0.14 ± 0.03 ab 0.47 ± 0.02 a*
0.26 ± 0.02 b 0.12 ± 0.01 ab 0.37 ± 0.02 b
0.25 ± 0.02 b 0.11 ± 0.01 b 0.36 ± 0.02 b
0.002 0.020 0.001
by these genes. We suggest that transcriptomic analyses
should be performed in order to know this. It was deter-
mined that many of the gene clusters that are present in the
reference strain B. amyloliquefaciens FZB42, active prin-
ciple of Biomex® and Rhizovital®42, are present in
B. subtilis EA-CB0575; this makes this strain promissory
for bioproducts development. The topologies of the phylo-
genetic trees obtained were coincident and have high
values of support in most branches (BP, > 75; PP, > 0.95).
It was determined that the offered resolution by trees based
on the 16S gene is not enough to differentiate the strains in
the B. amyloliquefaciens, B. subtilis, and B. licheniformis
groups, probably because of the high similarity in 16s
rDNA sequences (Rooney et al. 2009). Two clades were
found: one for B. thuringiensis, B. anthracis, B. cereus,
B. mycoides, and B. weihenstephanensis, considered path-
ogens of several hosts; and a second clade formed by
B. pumilus, B. licheniformis, B. subtilis, B. velezensis,
and B. amyloliquefaciens, all present in the soil.
B. amyloliquefaciens, B. velezensis, and B. subtilis differ
in well-resolved siblings clades and independent of
B. licheniformis clade, which did not happen in phylogeny
inferred with information from the 16S ribosomal gene
(Fig. S4).
We could determine the PGPR potential of B. subtilis EA-
CB0575 and B. subtilis 168 strains in tomato plants. We eval-
uated this potential for B. subtilis EA-CB075 in others crops
as banana (Posada et al. 2016), and we established a potential
colonization capacity on tomato and banana roots using FISH
and CARD-FISH methods (Posada et al. 2018). These results
are related with bacterial genomic and biochemical potential
evaluated in this research. Better performance of B. subtilis
microorganisms than B. velezensis to promote plant TDW was
found with percentages from 25.40 to 34.60%. B. velezensis
did not show differences with control; however, this strain has
been reported as a growth promoter through indirect promo-
tion mechanisms such as biocontrol (Chowdhury et al. 2015).
Funct Integr Genomics
Conclusion
B. subtilis EA-CB0575 is a potential PGPR due to its genomic
and phenotypic capacity related to the mechanisms of plant
growth promotion. Its genome was assembled in this project
in 16 contigs, and genes related to plant growth promotion
mechanisms were annotated and analyzed. It was found genes
related to IAA, siderophores, acetoin, 2,3-butanediol and LPs
production, nitrogen fixation, phosphate solubilization and
antagonistic activity against phytopathogens. The biochemi-
cal test allowed to determine the possibility of the microor-
ganism for the production of LPs of surfactin families
(surfacins C12, C13, C14, C15, and C16), fengycins A
(C14, C15, C16, C17), and fengycins B (C16 and C17).
Also, we corroborate its possibility for IAA and siderophores
production, to solubilize phosphates and fix environmental
nitrogen. It was determined that the microorganism has
3.9% of its genome dedicated to the production of secondary
metabolites and belongs to B. subtilis subtilis clade. In addi-
tion, B. subtilis EA-CB0575 and its type strains promote
TDW of the tomato plant.
Acknowledgments The authors acknowledge supercomputing resources
made available by the Centro de Computación Científica Apolo at
Universidad EAFIT (http://www.eafit.edu.co/apolo). This research was
made possible by the Subscribe Contract Numbers 166 and 139 from
2017 with the Ministerio de Medio Ambiente y Desarrollo Territorial in
the category “Contrato de Acceso a Recursos Genéticos y Productos
Derivados para la Investigación Científica” and “Contrato de Acceso a
Recursos Genéticos y Productos Derivados con Fines Comerciales,”
respectively.
Funding information This study was financially supported by the
Universidad EAFIT, the Association of Banana Growers of Colombia
(AUGURA) and Colciencias (contract number 0836-2012).
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